Many diseases of the retina and optic nerve, such as age-related macular degeneration, retinopathy of prematurity, diabetic retinopathy, and glaucoma, have been shown to involve reactive oxygen species (ROS). However, the complex biological functions of ROS in the retina are still unclear. A non-invasive in vivo monitoring technique of retinal ROS will be very helpful in further understanding of disease mechanisms related to these molecules. Recent studies have shown that L-012, a highly sensitive chemiluminescence probe, can be used to detect subcutaneous ROS activity by in vivo imaging. Hence, we will evaluate and optimize the application of in vivo L-012 chemiluminescence in detecting mouse retinal ROS after retinal ischemia/reperfusion (I/R), a study model well-known to induce high retinal ROS levels. Our objective in this application is to characterize and optimize the use of L-012 as a non-invasive in vivo method to measure ischemia-induced accumulation of ROS in the mouse retina. Our central hypothesis, based on our preliminary data, is that L-012 can reproducibly detect ROS in the retina and thus is a practical, non-invasive in vivo detection technology for retinal ROS levels. Our rationale for this study is that non-invasive in vivo detection method of retinal ROS is a very useful tool that will significantly aid our understanding of the involvement of oxidative damage in various retinopathies. Our long-term goal is to use this technology to study ROS-related biological changes and mechanisms in various retinopathies and apply this knowledge in designing therapeutic approaches for retinoprotection. Guided by strong preliminary data, we will test our hypothesis by pursuing two specific aims: 1) Determine if L-012 can be used to detect ROS accumulation in the retina after retinal I/R.; and 2) Determine if compounds that lower ROS level reduce the L-012 chemiluminescence after retinal I/R. Under Specific Aim #1, We plan to use non-invasive in vivo imaging to assess L-012 chemiluminescence in the eye of the same mouse at different time points after retinal I/R, and correlate these findings with post mortem biochemical and immunohistochemical detection of oxidative changes in the retina. Under Specific Aim #2, we will treat mice with compounds that interfere with ROS levels at different time points after I/R. We will then evaluate and correlate L-012 chemiluminescence with biochemical and immunohistochemical oxidative changes in the retina. This project is significant because it will establish, characterize, and optimize a new method for practical, non-invasive, in vivo real-time evaluation of retinal ROS level. This project is novel because this study will identify a novel, non-invasive technique for in vivo assessment of ROS levels in retina, which will improve the temporal and quantitative understanding of this important biological function. This novel understanding will aid in the eventual design of therapeutic treatment for retinal diseases.